Inflammatory responses are defensive responses which are caused by various factors such as infection with pathogens or injury of tissue in the body and perform initial protective action to limit damage to infected or injured areas. In most cases, such inflammatory responses lead to the removal of pathogenic factors and the induction of adaptive immunity by the components of innate immunity (Hawiger J., Innate Immunity and Inflammation: A Transcriptional Paradigm. Immunologic Research. 23, pp. 99-109, 2001). Rubors, tumors, calors, dolors and the like, known to be accompanied by inflammation, are the results of continuous inflammatory responses, such as an increase in local blood flow and a decrease in local blood flow rate, which result from vasodilation caused by the action of inflammatory mediators and cytokines in the area of inflammation, an increase in the release of plasma components from blood vessels, which results from an increase in the permeability of blood vessels, an increase in the release of immune cells from blood vessels, which results from the adhesion of blood vessel endothelial cells to circulating immune cells, and an increase in migration to an infected area by chemotaxis (Gallo R L, Murakami M, Takaaki O, Zaiou M., Biology and clinical relevance of naturally occurring antimicrobial peptides. J. Allergy. Clin. Immunol. 110, pp. 823-831. 2002; Graeme B. Ryan, M B, and Guido M., Acute Inflammation. American Journal of Pathology. 86(1), pp. 185-274, 1977). In addition, inflammation-related vasodilation and an increase in the permeability of blood vessels are also attributable to histamines which are produced in some cells in response to tissue injury, and low-molecular-weight peptide kinins which are present in an inactivated state in blood and activated by tissue injury (Yamaki K, Thorlacius H, Xie X, Lindbom L, Hedqvist P, Raud J., Characteristics of histamineinduced leukocyte rolling in the undisturbed microcirculation of the rat mesentry. British J. Pharmacol. 123, pp. 390-399, 1998; Brocklehurst W E, Role of Kinins and Prostaglandins in Inflammation. Proc. Roy. Soc. Med. 64, pp. 4-6, 1971).
Generally, an acute inflammatory response occurs rapidly, is maintained for a short time and is accompanied by a systemic response known as the acute-phase response. Meanwhile, chronic inflammation can result continuous immune activation in connection with some diseases such as infection or autoimmune diseases, and the accumulation and activation of macrophages is the hallmark of chronic inflammation (Huang A L, Vita J A, Effects of Systemic Inflammation on Endothelium-Dependent Vasodilation. Trends, Cardiovasc. Med. 16, p. 1520, 2006). However, continuous chronic inflammatory responses can cause serious damage to host cells or tissue.
Inflammatory responses at the site of infection are initiated by the response of macrophages to pathogens. It is known that reactive oxygen species and reactive nitrogen species (e.g., NO), which are produced by macrophages activated by pathogens, inflammatory mediators such as prostaglandins and leukotrienes, and pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8, are involved in inflammatory responses (Renauld J C, New insights into the role of cytokines in asthma. J. Clin. Pathol. 54, pp. 577-589, 2001; Blake G J, Ridker P M, Tumour necrosis factor-α, inflammatory biomarkers, and atherogenesis. Eur. Heart J. 23, pp. 345347, 2002). The activation of NF-κB that is a transcriptional factor of genes related to the production of inflammatory mediators is very important in the inflammation-related action of macrophages. It was reported that inflammation-related genes, including inducible nitric oxide synthase (iNOS2), cyclooxygenase (COX-2), TNF-α, IL-6, IL-8 and the like, are transcribed by NF-κB in macrophages.
Nitric oxide (NO) is produced from L-arginine by nitric oxide synthase (hereinafter referred to as NOS) in the macrophages of the human body (Kerwin, J. F. et al., J. Med. Chem., 38:4343, 1995). The NOS of the human body has three NOS isomers, including endothelial constitutive NOS (hereinafter referred to as ecNOS), neuronal constitutive NOS (hereinafter referred to as ncNOS) and inducible NOS (hereinafter referred to as iNOS). Among them, ecNOS and ncNOS are expressed in endothelial cells and neuronal cells, respectively, and dependent on calcium and calmodulin, whereas iNOS is highly expressed in various cells only when the cells are activated by lipopolysaccharide (hereinafter referred to as LPS) present in the cell membrane of pathogenic bacteria, cytokines such as IL-1 and TNF-α, and immune stimulants such as radiation, and iNOS is not dependent on calcium and calmodulin. NO functions as a defense against tumor cells and pathogenic bacteria at high concentration, and a low concentration of NO produced in blood vessel endothelial cells functions to regulate blood pressure, and NO produced in neuronal cells performs various physiological responses related to neurotransmitter function, learning, memory and the like. Constitutive NOS (cNOS) plays an important role in maintaining the homeostasis of the human body, and NO produced by ecNOS acts in the blood vessel system to inhibit vasodilation and platelet adhesion or aggregation, and NO produced by ncNOS acts in the nerve system to increase long-term memory or acts as a neurotransmitter to cause melancholia and is involved in the mobility of the digestive tract or the erection of the penis.
On the other hand, NO produced by iNOS expression induced by specific cytokines or LPS is involved in inflammatory expression or host defense mechanisms. The transcriptional factor NF-κB is activated in macrophages by the stimulation of endotoxin to induce iNOS expression, thereby increasing the production of NO (Butler, A. R., Chemistry & Industry, 16:828, 1995). It is known that when the expression of iNOS is induced by external stimulation of LPS, inflammation inducers radiation or the like, a large amount of NO is produced continuously for 4-6 hours to cause inflammatory responses in the human body. In the case of rats, large amounts of iNOS mRNA and protein are expressed in macrophages by external stimulation, and NO synthesized by the expression performs antimicrobial action and antitumor action. However, it is known that when NO is produced in excessively large amounts, it causes inflammatory responses, such as arthritis and septicemia, tissue graft rejection, immune diseases such as autoimmune diseases and diabetes, and the death of neuronal cells.
Thus, iNOS activity inhibitors have high potential as agents for treating such diseases, and from this viewpoint, compounds that inhibit NO production caused by iNOS can be used as agents for treating various inflammatory diseases in the human body. Studies on materials that inhibit the production of NO have been conducted mainly on the development of materials that specifically inhibit the enzymatic activity of iNOS. Specifically, studies have been conducted on the development of derivatives of the precursor L-arginine, derivatives of L-citrulline, derivatives of amino guanidine, derivatives of isothiourea, etc., (Babu, B. R. B. and Griffith O. W., Current Opinion in Chemical Biology, 2:491, 1998).
However, the regulation of expression of iNOS in the transcription stage in inflammatory responses is very important in determining the production of NO, and thus there is a need for the development of drugs related to the regulation of NF-κB activity by phosphorylation of IkB which is an inhibitor of the transcriptional factor NF-κB in macrophages, or drugs related to Akt, ERK, c-jun- and p38-MAPK signaling pathways which are related to the regulation of NF-κB activity.
Lichens are similar to non-flowering plants and are the symbiotic association of fungi (mycobionts) with algae and/or cyanobacteria (photobionts). The fungi in lichens form thalli or lichen substrates containing typical secondary metabolites (Ahmadjin V., The lichen symbiosis, Wiley, New York, pp. 1-6, 1993). It is difficult to obtain sufficient amounts of natural lichen samples, and technology of cultivating large amounts of lichens is not known. For this reason, studies on lichens were relatively insufficient compared to studies on higher plants. As the tissue culture method, mass-production method and biological analysis method for lichens have been improved, studies thereon have been actively conducted (Behera, B. C. et al., Lebensm. Wiss. Technol., 39:805, 2006). Compounds having various biological activities (including cytotoxicity, antifungal, antimicrobial, antioxidant and anti-inflammatory activities), including fatty acids, depsides, depsidones, dibenzofurans, diterpenes, anthraquinones, naphtoquinones, usninic acid, pulvinic acids, xanthones and epidithiopiperazinediones, were isolated from lichens (Muller, K., Appl. Microbiol. Biotechnol., 56:9-16, 2001).
Ramalina terebrata is a lichen that grows naturally in the Antarctic King George Island and can be easily collected from the King George Island. During studies on the Antarctic lichen Ramalina terebrata, the present inventors previously isolated the novel compound Ramalin having excellent antioxidant activity (Korean Patent Application No. 2008-111021). However, there has been no report that Ramalin has anti-inflammatory activity.
Accordingly, the present inventors have examined the cellular immunity inhibitory activity of Ramalin by conducting comparative experiments in vitro and in vivo in order to determine the effect of Ramalin on the LPS-induced production of NO in mouse macrophage RAW264.7 cells, and as a result, have found that Ramalin has the effect of inhibiting inflammatory responses. More specifically, the present inventors have found that Ramalin of the present invention inhibited the mRNA expression of iNOS to significantly inhibit the production of NO, inhibited the activation of NF-κB, inhibited p38 MAPK, ERK1/2 and JNK signaling pathways, inhibited the expression of the LPS receptor TLR4, and showed excellent anti-inflammatory and immune regulatory effects in in vivo experiments, thereby completing the present invention.